PROJECT SUMMARY The ClC family of chloride channels and transporters play important physiological roles. Mutations in ClC family proteins are associated with an array of diseases, some of which are caused by protein misfolding. Thus, it is important to understand how these protein fold. Yet the folding of ClC family proteins presents a formidable challenge, for both biology and experiment. Not only are they large membrane proteins, but they have a remarkably complex topology with many helices that insert only part way into the bilayer and then loop back out, leaving considerable polar surfaces in the center of the bilayer. How proteins like this can fold in the apolar bilayer remains unknown and largely unexamined. Using a new single molecule forced unfolding technique we developed in the prior grant period, we made some surprising discoveries regarding the folding of the ClC antiporter from E. coli that we now propose to investigate more deeply. In particular, it appears that the protein folds in two domains that would require plunging polar moieties into the apolar bilayer. Why would the protein be designed to fold in this manner and how might it be accomplished? To address these issues we propose the following aims: Aim I: Structure of isolated ClC domains. We will investigate the structures of the domains with polar exposed surfaces in isolation and how they adapt to the bilayer environment. Aim II: How do lipid properties affect the stability of the isolated domains? To gain insight into the interaction of the protein and lipids during unfolding and folding, we will examine the effects of different lipids on the energetics of domain separation. Aim III: Can separate folding of the domains increase folding efficiency? Is there evidence for N- to C-terminal folding? We hypothesize that dividing the folding of ClC-ec into two units simplifies the folding of such a large complex protein. As the protein emerges from the translocon, the N-terminal domain may commence folding, reducing options for the C-terminal domain and possibly templating C-terminal domain folding. To test this hypothesis we will examine the folding efficiency and folding kinetics of the individual domains by themselves and in the presence of the other domain. Our work will map the folding energetics and pathway for arguably the most complex protein ever studied at this level; provide insight into the folding of the large number of membrane proteins with re-entrant helices; and provide new ideas for intervening in membrane protein folding diseases.